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Actions of Ba and Sr Ions on Isolated Rat Ileum
ACTIONS
OF
Kohtaro
Ba
AND
TANIYAMA,
Sr IONS
Nobuyoshi
ON
ISOLATED
YOSHIDA,
and Hiromasa
Nobuyuki
RAT
ILEUM
TAKAHASHI
ARAKI
Deparimeni of Pharnnacologe, Kohe University School of Medicine, Ikuta-ku, Kohe 650, Japan Accepted November 29, 1976
it is generally accepted that Ca ion is an essential substance in the contractile response of smooth muscle.
There are several reports that action of the Sr ion resembles that of the
Ca ion on smooth muscle.
Such include the stabilizing action on cell membrane (1, 2) and
the direct stimulation to muscle contractile elements to elicit contraction (1, 3, 4).
On the
other hand, it has been reported that the Ba-induced contraction is dependent on the mobili zation of Ca (5-7), that Ba activates contractile proteins (8) or that Ba produces a contraction by these two actions (9).
In an attempt to elucidate the discrepancies, actions of Ba and
Sr ions were investigated in relation to mobilization of the Ca ion. Male rats (200-250 g) were sacrificed by a blow on the neck, the ileum was removed and dissected, and strips approx. 1.5 cm long, were fixed vertically with a load of 0.5 g in the preparation bath containing 30 ml of nutrient solution (Locke's solution: NaCI 154, KCI 5.6, CaCl2 2.2, glucose 5.6, NaHCO3 4.8 (mM)). The medium was maintained at 27_f_1 °C and continuously bubbled with a mixture of 95 i,' 02 and 5 % CO2. Muscle contractions were isotonically recorded on a smoked paper with an approx. lOX magnification.
Pre
parations were allowed to equilibrate for 90 min in normal media, before experiments were begun. Ca-free solution was prepared in the same fashion as Locke's solution except that CaCI, was omitted and 0.01 mM GEDTA was added.
K-depolarizing Ca-free solution
was prepared in the same way as the Ca-free solution except that 56 mM Na was replaced by an equiamount of K. Ba (0.5-10 mM) produced a sustained contraction in normal medium.
The contraction
by Ra (0.5-10 mM) was not modified after a 3 min exposure to Ca-free medium, whereas it gradually decreased by repeated treatments with Ba at an interval of 30 min in Ca-free medium and it then remained at a constant magnitude after approx. 300 min exposure to Ca-free medium, when the K-induced contraction had disappeared.
On the other hand,
Sr (2--10 mM) produced a sustained relaxation in normal medium, whereas after a 3 min exposure to Ca-free medium, Sr (0.5-10 mM) caused a sustained contraction.
The contrac
tion by Sr (0.5-10 mM), like that by Ba, was gradually decreased by repeated treatments with Sr at an interval of 30 min in Ca-free medium and remained at a constant magnitude approx. 300 min after Ca removal.
These contractions by Ba and Sr were not modified by
5'/1()-4 mM atropine, which abolished acetylcholine-induced maximal contraction.
Thus,
the contractions by Ba and Sr were the result of a release of Ca from storage sites, until a constant magnitude; was attained in th.. Ca-free medium.
In the following experiments,
Fa . I. Effect of Ba (a) and Sr (b) on the concentration-action curves of Ca obtained in K-depolarizing Ca-free medium. Cumulative concentration-action curves of Ca were prepared after contractions by Ba and Sr had remained at a constant magnitude in Ca-free medium. Magnitude of maximal contraction by Ca was taken as 100%. •-0, control; in the presence of 1 mM Ba or Sr; ~~ in the presence of 5 mM Ba or Sr, ;< concentration-action curve of Ba or Sr. Vertical bars represent S.E. No. of preparations, 8-18.
preparations
in which the contractions
in Ca-free (0.2-10
media,
mM),
produced
were exposed
Ba (0.5-10
sustained
were obtained
by Ba and Sr had remained
to K-depolarizing
mM) or Sr (0.5-10
contractions.
Ca-free
tively.
On the other hand,
left in low concentrations
As shown
of Ca and to the right in high concentrations
interaction
magnitude
(Fig. 1).
agonist,
act on the same activities
where
Sr.
receptor
by
of the occupancy
theory in
each other at the point located
of Sr at the
by Sri the effects of 1 mM and 5 mM Sr on the C-A curve
site, our findings constants
dualism
where a'
of Ca.
may
(10) between full agonist,
be interpreted
with receptors
Ca,
Sri are assumed
as follows:
to
the intrinsic
of Ca and Sr are represented
,3, and the total number
of Ca, YCa, is formulated
[Ca] is the concentration
formulated
of Ca by pretreatments
If the full agonist, Ca, and the partial agonist,
respectively,
The activity
of Ca
Since the log C-A Curves of Ca in the absence
to the theory of competitive
and the association
,3, Kca and Ks, by Rt.
(10, Il).
of maximal contraction
and partial
curves
1-(a), the log C-A
These shifts of the C-A curves
interaction
of 1 mM and 5 mM Sr crossed
of Ca may correspond
(C-A)
in Fig.
medium
for 10 min with 1 mM and 5 in M Ba, respec
of Ca by Ba and Sr are similar to the competitive
and in the presence
Ca-free
of Ca
as shown in Fig. 1-(b), the log C-A curves of Ca shifted to the
for 10 min with 1 mM and 5 mM Sri respectively
drug-receptor
magnitude
The addition
concentration-action
medium.
curves of Ca shifted to the left by pretreatments
at a constant
medium.
mM) to the K-depolarizing
Cumulative
in K-depolarizing
Ca-free
of receptors
by a,
is expressed
by
The dualistic
activity
of Ca and
Sri YCa , Sri is
where [Sr] is the concentration
of Sr.
From the formulae
(1) and (2), the pair of coordinates
ofthecrossing point between logC-Acurve ofY(,,, andthatofY(,a Sris( 3 (cr 3)Kcs, ,iRt)' That is, the crossing point is shown to be located at the height of maximal contraction induced
by Sr.
Thus,
ating on Ca-receptor,
Sr shows competitive contractile
proteins.
extent, present in the tissue in which Sr-induced in the Ca-free medium. coil (12), therefore,
tissue Ca may participate
to the theory
agonist, Ba.
of competitive
Ebashi (I) reported
show From
from storage replace
synergism
intracellular
magnitude
by Sr in the rat ileum.
synergism it appears
sites to elicit contractions.
full agonist,
On
Ca, and partial
of myosin B to Ba ion of barnyard
proteins,
to Ca-induced that
by binding
antagonize
competitively
Since
may also be low in rat ileum, contraction,
not
Ba and Sr ions induce
In the absence
contraction
fo~~I
the affinity of Ba for the contractile
muscle of guinea pig taenia coli (13).
contractile
a competitive
Sr ion would
at a constant
to be the case in guinea pig taenia
(10) between
Furthermore,
these findings,
Ca in generating
(3 Rt) by oper
that Ca ion is, to some
remained
in the contraction
is lower than that of Sr in glycerinated
dualism.
it is possible
contraction
that the sensitivity
the affinity of Ba for the Ca-receptor, Ba would
However,
contractions
of I mM and 5 mM Ba on the log C-A Curve of Ca may cor
gizzard was lower than that to Sr ion. protein
to Ca-induced
Such has already been suggested
the other hand, the actions respond
dualism
of external
to contractile the binding
a competitive a release of Ca
Ca, these ions would
proteins.
Furthermore,
of Ca ion to contractile
proteins. REFERENCES I) EBASHI,S.: Prof. 4th Intel-national Congress oil Pharmacology, Edited by EIGENMANN,R., Vol. 1, p. 32, Schwabe and Co., Basel and Stuttgart (1970); 2) HOTTA, Y. AND TsuKUI, R.: Nature 217, 867 (1968): 3) DANIEL, E.E., SEHDEV,H. AND ROBINSON,K.: Physiol. Rev. 5, 228 (1962); 4) EBASHI,S., EBASHI,F. AND KODAMA,A.: J. Biochen. 62, 137 (1967); 5) KARAKI, H., IKEDA,M. AND URAKAWA,N.: Japall. J. Pharmacol. 17, 496 (1967): 6) NORTHOVER,B.J.: Brit. J. Pharmacol. 34, 417 (1968); 7) SPERELAKIS, N.: Fedn Proc. 22, 461 (1963); 8) SCHILD, H.O.: Brit. J. Pharmacol. 31, 578 (1967): 9) DANIEL,E.E.: A. Rev. Pharmacol. 4, 189 (1964); 10) ARII:NS, E.J., VAN ROSSUM, J.M. .vND SIMONIs, A.M.: Ar_neinl.-Forsch. 6, 282 (1956): 11) MATSUMOTO, H.: Seikagakitteki Ya/ur eaklt, Edited by YOSHIDA,H. AND TANAKA,S., p. 73, Asakura Shoten, Tokyo (1971) (ill Japanese); 12) NASU, T. AND URAKAWA,N.: Japan. J. Pharmacol. 24, 543 (1974); 13) BANDO, T., Aizu, M., SAKATO,M. AND YANAGISAWA,M.: Folia pharmacol. japon. 66, 89 (1970) (in Japanese)
OF
Kohtaro
Ba
AND
TANIYAMA,
Sr IONS
Nobuyoshi
ON
ISOLATED
YOSHIDA,
and Hiromasa
Nobuyuki
RAT
ILEUM
TAKAHASHI
ARAKI
Deparimeni of Pharnnacologe, Kohe University School of Medicine, Ikuta-ku, Kohe 650, Japan Accepted November 29, 1976
it is generally accepted that Ca ion is an essential substance in the contractile response of smooth muscle.
There are several reports that action of the Sr ion resembles that of the
Ca ion on smooth muscle.
Such include the stabilizing action on cell membrane (1, 2) and
the direct stimulation to muscle contractile elements to elicit contraction (1, 3, 4).
On the
other hand, it has been reported that the Ba-induced contraction is dependent on the mobili zation of Ca (5-7), that Ba activates contractile proteins (8) or that Ba produces a contraction by these two actions (9).
In an attempt to elucidate the discrepancies, actions of Ba and
Sr ions were investigated in relation to mobilization of the Ca ion. Male rats (200-250 g) were sacrificed by a blow on the neck, the ileum was removed and dissected, and strips approx. 1.5 cm long, were fixed vertically with a load of 0.5 g in the preparation bath containing 30 ml of nutrient solution (Locke's solution: NaCI 154, KCI 5.6, CaCl2 2.2, glucose 5.6, NaHCO3 4.8 (mM)). The medium was maintained at 27_f_1 °C and continuously bubbled with a mixture of 95 i,' 02 and 5 % CO2. Muscle contractions were isotonically recorded on a smoked paper with an approx. lOX magnification.
Pre
parations were allowed to equilibrate for 90 min in normal media, before experiments were begun. Ca-free solution was prepared in the same fashion as Locke's solution except that CaCI, was omitted and 0.01 mM GEDTA was added.
K-depolarizing Ca-free solution
was prepared in the same way as the Ca-free solution except that 56 mM Na was replaced by an equiamount of K. Ba (0.5-10 mM) produced a sustained contraction in normal medium.
The contraction
by Ra (0.5-10 mM) was not modified after a 3 min exposure to Ca-free medium, whereas it gradually decreased by repeated treatments with Ba at an interval of 30 min in Ca-free medium and it then remained at a constant magnitude after approx. 300 min exposure to Ca-free medium, when the K-induced contraction had disappeared.
On the other hand,
Sr (2--10 mM) produced a sustained relaxation in normal medium, whereas after a 3 min exposure to Ca-free medium, Sr (0.5-10 mM) caused a sustained contraction.
The contrac
tion by Sr (0.5-10 mM), like that by Ba, was gradually decreased by repeated treatments with Sr at an interval of 30 min in Ca-free medium and remained at a constant magnitude approx. 300 min after Ca removal.
These contractions by Ba and Sr were not modified by
5'/1()-4 mM atropine, which abolished acetylcholine-induced maximal contraction.
Thus,
the contractions by Ba and Sr were the result of a release of Ca from storage sites, until a constant magnitude; was attained in th.. Ca-free medium.
In the following experiments,
Fa . I. Effect of Ba (a) and Sr (b) on the concentration-action curves of Ca obtained in K-depolarizing Ca-free medium. Cumulative concentration-action curves of Ca were prepared after contractions by Ba and Sr had remained at a constant magnitude in Ca-free medium. Magnitude of maximal contraction by Ca was taken as 100%. •-0, control; in the presence of 1 mM Ba or Sr; ~~ in the presence of 5 mM Ba or Sr, ;< concentration-action curve of Ba or Sr. Vertical bars represent S.E. No. of preparations, 8-18.
preparations
in which the contractions
in Ca-free (0.2-10
media,
mM),
produced
were exposed
Ba (0.5-10
sustained
were obtained
by Ba and Sr had remained
to K-depolarizing
mM) or Sr (0.5-10
contractions.
Ca-free
tively.
On the other hand,
left in low concentrations
As shown
of Ca and to the right in high concentrations
interaction
magnitude
(Fig. 1).
agonist,
act on the same activities
where
Sr.
receptor
by
of the occupancy
theory in
each other at the point located
of Sr at the
by Sri the effects of 1 mM and 5 mM Sr on the C-A curve
site, our findings constants
dualism
where a'
of Ca.
may
(10) between full agonist,
be interpreted
with receptors
Ca,
Sri are assumed
as follows:
to
the intrinsic
of Ca and Sr are represented
,3, and the total number
of Ca, YCa, is formulated
[Ca] is the concentration
formulated
of Ca by pretreatments
If the full agonist, Ca, and the partial agonist,
respectively,
The activity
of Ca
Since the log C-A Curves of Ca in the absence
to the theory of competitive
and the association
,3, Kca and Ks, by Rt.
(10, Il).
of maximal contraction
and partial
curves
1-(a), the log C-A
These shifts of the C-A curves
interaction
of 1 mM and 5 mM Sr crossed
of Ca may correspond
(C-A)
in Fig.
medium
for 10 min with 1 mM and 5 in M Ba, respec
of Ca by Ba and Sr are similar to the competitive
and in the presence
Ca-free
of Ca
as shown in Fig. 1-(b), the log C-A curves of Ca shifted to the
for 10 min with 1 mM and 5 mM Sri respectively
drug-receptor
magnitude
The addition
concentration-action
medium.
curves of Ca shifted to the left by pretreatments
at a constant
medium.
mM) to the K-depolarizing
Cumulative
in K-depolarizing
Ca-free
of receptors
by a,
is expressed
by
The dualistic
activity
of Ca and
Sri YCa , Sri is
where [Sr] is the concentration
of Sr.
From the formulae
(1) and (2), the pair of coordinates
ofthecrossing point between logC-Acurve ofY(,,, andthatofY(,a Sris( 3 (cr 3)Kcs, ,iRt)' That is, the crossing point is shown to be located at the height of maximal contraction induced
by Sr.
Thus,
ating on Ca-receptor,
Sr shows competitive contractile
proteins.
extent, present in the tissue in which Sr-induced in the Ca-free medium. coil (12), therefore,
tissue Ca may participate
to the theory
agonist, Ba.
of competitive
Ebashi (I) reported
show From
from storage replace
synergism
intracellular
magnitude
by Sr in the rat ileum.
synergism it appears
sites to elicit contractions.
full agonist,
On
Ca, and partial
of myosin B to Ba ion of barnyard
proteins,
to Ca-induced that
by binding
antagonize
competitively
Since
may also be low in rat ileum, contraction,
not
Ba and Sr ions induce
In the absence
contraction
fo~~I
the affinity of Ba for the contractile
muscle of guinea pig taenia coli (13).
contractile
a competitive
Sr ion would
at a constant
to be the case in guinea pig taenia
(10) between
Furthermore,
these findings,
Ca in generating
(3 Rt) by oper
that Ca ion is, to some
remained
in the contraction
is lower than that of Sr in glycerinated
dualism.
it is possible
contraction
that the sensitivity
the affinity of Ba for the Ca-receptor, Ba would
However,
contractions
of I mM and 5 mM Ba on the log C-A Curve of Ca may cor
gizzard was lower than that to Sr ion. protein
to Ca-induced
Such has already been suggested
the other hand, the actions respond
dualism
of external
to contractile the binding
a competitive a release of Ca
Ca, these ions would
proteins.
Furthermore,
of Ca ion to contractile
proteins. REFERENCES I) EBASHI,S.: Prof. 4th Intel-national Congress oil Pharmacology, Edited by EIGENMANN,R., Vol. 1, p. 32, Schwabe and Co., Basel and Stuttgart (1970); 2) HOTTA, Y. AND TsuKUI, R.: Nature 217, 867 (1968): 3) DANIEL, E.E., SEHDEV,H. AND ROBINSON,K.: Physiol. Rev. 5, 228 (1962); 4) EBASHI,S., EBASHI,F. AND KODAMA,A.: J. Biochen. 62, 137 (1967); 5) KARAKI, H., IKEDA,M. AND URAKAWA,N.: Japall. J. Pharmacol. 17, 496 (1967): 6) NORTHOVER,B.J.: Brit. J. Pharmacol. 34, 417 (1968); 7) SPERELAKIS, N.: Fedn Proc. 22, 461 (1963); 8) SCHILD, H.O.: Brit. J. Pharmacol. 31, 578 (1967): 9) DANIEL,E.E.: A. Rev. Pharmacol. 4, 189 (1964); 10) ARII:NS, E.J., VAN ROSSUM, J.M. .vND SIMONIs, A.M.: Ar_neinl.-Forsch. 6, 282 (1956): 11) MATSUMOTO, H.: Seikagakitteki Ya/ur eaklt, Edited by YOSHIDA,H. AND TANAKA,S., p. 73, Asakura Shoten, Tokyo (1971) (ill Japanese); 12) NASU, T. AND URAKAWA,N.: Japan. J. Pharmacol. 24, 543 (1974); 13) BANDO, T., Aizu, M., SAKATO,M. AND YANAGISAWA,M.: Folia pharmacol. japon. 66, 89 (1970) (in Japanese)